This invention relates to nucleic acid sequences encoding 1-deoxy-D-xylulose-5-phosphate reductoisomerase.
Isoprenoids are a large and structurally diverse group of compounds that play essential roles in plants as hormones, photosynthetic pigments, electron carriers, and components of membranes, and that also serve in communication and defense (Harborne, J. B. (1991) in Ecological Chemistry and Biochemistry of Plant Terpenoids (Harborne, J. B., and Tomas-Barberan, R. A., Eds.), pp. 399-426. Clarendon Press, Oxford). Until recently, it was widely accepted that all isoprenoids were synthesized via the acetate/mevalonate pathway (Spurgeon. S. L., and Porter, J. W. (1983) in Biosynthesis of Isoprenoid Compounds (Porter, J. W., and Spurgeon, S. L., Eds.), Vol. 1, pp. 1-46, John Wiley, N.Y.).
However, evidence has emerged over the last few years that isopentenyl diphosphate, the central intermediate of isoprenoid biosynthesis, originates from pyruvate and D-glyceraldehyde-3-phosphate via a new mevalonate-independent pathway in several eubacteria (Rohmer, M., et al., Biochem. J. 295, 517-524 (1993); Broers, S. T. J. (1994) Ph.D. Thesis, Eidgenxc3x6ssische Technische Hochschule, Zxc3xcrich, Switzerland; Rohmer, M., et al., J Am. Chem. Soc. 118, 2564-2566 (1996)), algae (Schwender, J., et al., Biochem. J. 316, 73-80 (1996) ), and plant plastids (Schwarz, M. K. (1994) Ph.D. Thesis, Eidgenxc3x6ssische Technische Hochschule, Zxc3xcrich, Switzerland; Lichtenthaler, H. K., et al., FEBS Lett. 400, 271-274 (1997)). The first step in this novel pathway involves a transketolase-type condensation reaction of pyruvate and glyceraldehyde-3-phosphate to yield 1-deoxy-D-xylulose-5-phosphate (FIG. 1). Genes encoding the enzyme which catalyzes this reaction, deoxyxylulose phosphate synthase, have been cloned from E. coli (Sprenger, G. A., et al., Proc. Natl. Acad Sci. USA 94, 12857-12862 (1997); Lois, L. M. et al., Proc. Natl. Acad. Sci. USA 95, 2105-2110 (1998)), peppermint (Mentha x piperita) (Lange, B. M. et al., Proc. Natl. Acad Sci. USA 95, 2100-2104 (1998)) and pepper (Bouvier, F. et al., Plant Physiol. 117, 1423-1431 (1998)).
The second step of the mevalonate-independent pathway is considered to involve an intramolecular rearrangement and subsequent reduction of deoxyxylulose phosphate to yield 2-C-methyl-D-erythritol4-phosphate (Duvold, T. et al., Tetrahedron Lett. 38, 4769-4772 (1997); Duvold, T. et al., Tetrahedron Lett. 38, 6181-6184 (1997); Sagner, S. et al., Tetrahedron Lett. 39, 2091-2094 (1998)) (FIG. 1). Seto and coworkers (Takahashi, S. et al., Proc. Natl. Acad. Sci. USA 95, 9879-9884 (1998)) have recently reported the isolation and characterization of a reductoisomerase gene from E. coli. The present invention provides a nucleic acid molecule isolated from peppermint that encodes a 1-deoxy-D-xylulose-5-phosphate reductoisomerase.
In accordance with the foregoing, a cDNA encoding a 1-deoxy-D-xylulose-5-phosphate reductoisomerase from peppermint (Mentha piperita) has been isolated and sequenced, and the corresponding amino acid sequence has been deduced. Accordingly, the present invention relates to isolated DNA sequences which code for the expression of plant 1-deoxy-D-xylulose-5-phosphate reductoisomerase, such as isolated DNA sequences which code for the expression of 1-deoxy-D-xylulose-5-phosphate reductoisomerase from essential oil plants, including plants of the genus Mentha. A representative example of an isolated, Mentha DNA sequence which codes for the expression of 1-deoxy-D-xylulose-5-phosphate reductoisomerase is set forth in SEQ ID NO:1 which encodes a 1-deoxy-D-xylulose-5-phosphate reductoisomerase protein (SEQ ID NO:2) from peppermint (Mentha piperita). Additionally, the present invention relates to isolated plant 1-deoxy-D-xylulose-5-phosphate reductoisomerase proteins (including isolated 1-deoxy-D-xylulose-5-phosphate reductoisomerase proteins from essential oil plants, such as plants of the genus Mentha), including the peppermint (Mentha piperita) 1-deoxy-D-xylulose-5-phosphate reductoisomerase protein having the amino acid sequence set forth in SEQ ID NO:2.
In another aspect, the present invention relates to nucleic acid molecules that hybridize under stringent conditions to the nucleic acid molecule having the sequence set forth in SEQ ID NO:1, or to its complement, ie., to an antisense molecule that is complementary in sequence to the sequence set forth in SEQ ID NO:1. In other aspects, the present invention is directed to replicable recombinant cloning vehicles comprising a nucleic acid sequence, e.g., a DNA sequence which codes for a plant 1-deoxy-D-xylulose-5-phosphate reductoisomerase, or for a nucleotide sequence sufficiently complementary to at least a portion of DNA or RNA encoding a plant 1-deoxy-D-xylulose-5-phosphate reductoisomerase to enable hybridization therewith (e.g., antisense RNA or fragments of DNA complementary to a portion of DNA or RNA molecules encoding a plant 1-deoxy-D-xylulose-5-phosphate reductoisomerase which are useful as polymerase chain reaction primers or as probes for plant 1-deoxy-D-xylulose-5-phosphate reductoisomerase genes or related genes). In yet other aspects of the invention, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence of the invention.
Thus, the present invention provides for the recombinant expression of plant 1-deoxy-D-xylulose-5-phosphate reductoisomerase, and the inventive concepts may be used to facilitate the production, isolation and purification of significant quantities of recombinant 1-deoxy-D-xylulose-5-phosphate reductoisomerase (or of its primary enzyme products) for subsequent use, to obtain expression or enhanced expression of 1-deoxy-D-xylulose-5-phosphate reductoisomerase in plants, microorganisms or animals, or may be otherwise employed in an environment where the regulation or expression of 1-deoxy-D-xylulose-5-phosphate reductoisomerase is desired for the production of this enzyme, or its enzyme product, or derivatives thereof.